Chromosome Instability in Acute Myeloid Leukemia is associated to increased spontaneous breakage and Copy Number Variations (CNVs).

FUNDIA ARIELA; LARRIPA IRENE; ACEVEDO SUSANA

Nueva Orleans, USA

Congreso; 51st Annual Meeting of the American Society of Hematology; 2009

American Society of Hematology

Acute Myeloid Leukemia (AML) is a genetically pleiomorphic disease characterized by multiple genetic lesions including structural and numerical chromosome aberrations, gene mutations and changes in gene expression. The underlying mechanism behind the acquisition of these genetic abnormalities is not known, but it is likely that are facilitated by factors that increase chromosomal and genetic instability. The aim of this work was to explore chromosomal instability (CIN) in de novo AML patients by evaluating chromosome fragility and acquired DNA copy number variations (CNVs). Leukemic karyotypes were scored on bone marrow cultures (24-48 hs, without mitogens) from 24 AML patients using conventional cytogenetic and FISH. Spontaneous and FUdR -induced chromosomal fragility was studied on PHA-stimulated lymphocytes cultures (72 hs) from 10 patients and 10 healthy individuals, with and without FUdR (10mg/ml). One hundred metaphases were analyzed blind using conventional cytogenetic with sequential GTG banding. CNVs were scored on bone marrow cultures from 14 AML patients using fluorescence in situ hybridization (FISH) with two probes targeting specific regions on chromosomes 5q31 (LSI EGR/D5S721:D5S23) and 7q31 (LSI D7S522/CEP7), loci reported to be critical hotspots involved in AML. The cut-off value for FISH scoring was calculated after analyzing 250 cells from each of 10 normal bone marrow samples. The cutoff for allelic losses was equal to 0.8% for either 5q or 7q probe.Nine out of 24  AML cases presented normal karyotypes in bone marrow samples while the following abnormalities: t(3;8), t(3;11), t(4;11), t(15;17), t(9;22) and inv(16) were detected in the remaining patients. Significantly increased frequencies of spontaneous chromosome breakage, scored on untreated cultures, were detected in patients (0.22±0.03) respect to controls (0.07±0.03) (p<0.05), showing a random pattern of distribution. No differences were observed between patients and controls with FUdR treatment. Statistical analysis with Ch2 test considering data of FUdR breakpoint distribution over all individuals, identified 21 common fragile sites (c-fra) in AML cases (p<0.005). The most common sites were located at 1p32, 1p22, 1q21, 3p14, 3q27, 4q31, 5q31, 6p21 and 9q13. A high inter-individual variation in the pattern of expression was observed.Using FISH, we obtained the DNA copy numbers at 5q and 7q in all AML samples. Only losses of DNA were found with both probes. The 5q signal count in patients with normal karyotypes was on average 6.4% (range 0.5-30%), while it was 24.9% (range 0.8-90%) for patients with abnormal cytogenetic. The 7q signal count was on average 3.9% (range 0.5-10.3%) and 20.6% (range 2-63%) for cases with normal and abnormal karyotypes, respectively. Particularly, we showed that 12/14 (85.7%) AML cases, either with normal or abnormal karyotypes, exhibited copy number losses at both regions, showing different values between each patient. These findings showed that leukemic patients exhibit a CIN phenotype, providing an unstable background and facilitating the acquisition of additional genetic changes, such as CNVs, which could play an important role in disease progression.